Quantification of O2 by photoluminescence quenching is an established technique which has a number of attractive features, including reversible, non-chemical and non-invasive nature of sensing of O2. This methodology is actively used in various industrial and biomedical applications where measurement of O2 is required (Papkovsky D B. Meth. Enzymol. 2004, 381: 715-735).
Moreover, it is now recognised that in such samples the level of cell oxygenation can differ significantly from that of bulk medium. This may affect the cells and induce adaptive responses and physiologic changes. Hence there is a need to accurately monitor intracellular O2 levels and local O2 gradients in biological samples. Simple chemistries and measurement methodologies providing this are therefore important.
A number of photoluminescence based O2-sensing probes and techniques have been described so far. Rumsey W L et al. (Science, 1988, v. 241(4873): 1649-51), Dunphy, I, et al. (Anal Biochem 2002, 310(2): 191-8), Vinogradov S A et al. (U.S. Pat. No. 5,837,865, 1998), Wilson (U.S. Pat. No. 6,395,555, 2002), Hynes, J., et al. (J Biomol Screening, 2003, 8(3): 264-72), Cao Y. et al. (Analyst, 2004, 129(8): 745-50) describe probes for the sensing and imaging of O2 in systems containing live cells and tissue, and for the measurement of biological O2 consumption. These techniques normally employ O2-sensitive Pt- and Pd-porphyrins and some related complexes as probes, luminescence of which is quenched by O2. These probes were designed primarily for extracellular use, they are essentially cell-impermeable and can not be used for probing (intra)cellular O2.
Several probes including polymeric nanoparticles impregnated with oxygen-sensitive dyes (Koo Y E et al. Anal Chem, 2004, v. 76(9): 2498-505), lipobeads' (Ji J. et al. Anal Chem., 2001, 73(15): 3521-7); microspheres (Schmalzlin E., et al., Biophys. J. 2005, 89(2): 1339); hydrophilic metalloporphyrin dye bound to albumin (Howlett R A, J Appl Physiol. 2007, 102(4):1456-61) were applied to sensing intracellular O2 by loading the cells by microprojectile delivery, phagocytosis or microinjection (in large plant cells or skeletal muscle fibres). However, these systems have low loading efficiency, uneven distribution of the probe inside the cell, uncontrolled compartmentation and aggregation, significant cyto- and phototoxicity, they are rather complex and invasive.
Another approach to cells loading is the use special reagents which facilitate transport of O2 probes from the extracellular medium (O′Riordan T. C. et al. Anal. Chem., 2007 Dec. 15; 79(24):9414; Fercher a. et al.—Anal. Bioanal. Chem. 2010, Jan. 10, PMID: 20063150). It requires additional reagents and equipment, depends on the probe, cell, medium type and other conditions. In many cases, such loading is low, cell-specific and stressful, time-consuming and not very reproducible.
O2-sensitive probes with self (i.e. passive) loading capabilities were also described, for example those based on the conjugates of certain oxygen-sensitive dyes with cell-penetrating peptides (Neugebauer U, et al.—Chem. Comm., 2008 (42):5307-5309, Dmitriev RI et al.—Anal. Biochem. 398 (2010) 24-33). These molecular probes also have drawbacks. Thus, the former probe has low brightness and short fluorescence lifetime resulting in modest sensitivity to O2, and their utility in biological applications has not been demonstrated. The latter probes based on phosphorescent Pt- or Pd-coproporphyrins have modest photostability making them unusable in fluorescence oxygen imaging applications. Although these probes are brighter than Ru(II)-probes (higher molar absorptivity and emission yield), not always they provide sufficient signals for reliable and accurate O2 sensing experiments. Both probe types are limited to just a few dye structures, complex synthesis procedures and high costs.
It is an object of the invention to overcome at least one of the above-referenced problems.